Protein separation via ion-exchange chromatography and associated methods, systems, and devices

ABSTRACT

The present invention provides methods for separating proteins from a protein mixture. In one aspect, a method for separating a high concentration protein mixture into a bound protein fraction and a flow-through protein fraction can include delivering a protein mixture through an ion exchange column at a fixed pH and a fixed salt concentration. The fixed pH and the fixed salt concentration have been preselected to cause separation of the protein mixture into a bound protein fraction and a flow-through protein fraction. In this case, the bound protein fraction binds to the ion exchange column and the flow-though protein fraction flows though the ion exchange column. The method can further include receiving the flow-through protein fraction from the ion exchange column separate from the bound protein fraction, wherein either the bound protein fraction or the flow-through fraction contains a protein of interest.

PRIORITY DATA

This application is a continuation application of U.S. patentapplication Ser. No. 12/619,421, filed Nov. 16, 2009, now issued as U.S.Pat. No. 8,304,248.

FIELD OF THE INVENTION

The present invention relates to the separation and/or isolation ofproteins from a protein mixture. Accordingly, the present inventioninvolves the fields of medical diagnostics and biochemistry.

BACKGROUND OF THE INVENTION

The search for disease biomarkers has created a demand for rapid proteinanalysis from serum, plasma, and other complex protein mixtures. Severalseparation modalities, including chromatography, electrophoresis,isoelectric-focusing, and mass spectrometry are used in the search forbiomarkers for numerous disease states.

Serum and plasma, for example, contain a rich source of biomoleculesincluding a complex mixture of proteins. It is believed that manyproteins, including newly synthesized proteins and especially degradedprotein fragments, are transported in blood. Because of this, theconcentration of newly synthesized proteins or proteins that are beingdegraded may vary depending on circumstances. This can be especiallytrue in some disease states, thus potentially allowing diagnosis of suchdiseases through the presence of certain biomarkers. Serum, however,presents challenges for the physical detection of such proteins as theyare present at substantially lower concentrations as compared to highabundant proteins. Of the thousands of proteins that are present inblood, only a handful make up a vast majority of the total protein massin serum. Thus these “high abundant” proteins can cause interferenceproblems with most if not all protein separation methods.

One exemplary method that is often utilized to remove two of the highabundant proteins from serum, namely albumin and IgG, is based onimmunoaffinity chromatography. These affinity columns containimmobilized antibodies against human albumin and IgG that function tobind these proteins. The remainder proteins that do not, in theory, bindto the antibodies in the affinity column are retrieved in a pass-throughfraction. This method of extraction can be expensive and inefficient, asonly small amounts of serum (e.g. less than 50 μl) can be depleted ofalbumin and IgG on small columns (1 ml or less).

SUMMARY OF THE INVENTION

The present invention provides methods for separating proteins from aprotein mixture. In one aspect, for example, a method for separating ahigh concentration protein mixture into a bound protein fraction and aflow-through protein fraction includes delivering a protein mixturethrough an ion exchange column at a fixed pH and a fixed saltconcentration. The fixed pH and the fixed salt concentration have beenpreselected to cause separation of the protein mixture into a boundprotein fraction and a flow-through protein fraction. Thus, the boundprotein fraction binds to the ion exchange column and the flow-thoughprotein fraction flows though the ion exchange column. The methodfurther includes receiving the flow-through protein fraction from theion exchange column and then subsequently eluting the bound proteinfraction from the column and thus receiving the bound protein fractionseparate from the flow-through fraction. Additionally, either the boundprotein fraction or the flow-through fraction contains a protein ofinterest. Accordingly in one aspect, the protein of interest is in theflow-through protein fraction. In another aspect, the protein ofinterest is in the bound protein fraction.

Various protein mixtures are contemplated for use with the methodsaccording to aspects of the present invention. For example, in oneaspect, the protein mixture is a biological fluid. Non-limiting examplesof biological fluids can include blood serum, blood plasma, urine, CNSfluid, saliva, cellular extracts, tissue culture extracts, and mixturesthereof. Additionally, the protein mixture can be utilized in a varietyof forms. For example, in one aspect the protein mixture is an undilutedbiological fluid. In another aspect, the protein mixture is anon-dialyzed biological fluid. In yet another aspect, the proteinmixture is a non-ultafiltrated biological fluid. Furthermore, abiological fluid can be treated prior to separation. Non-limitingexamples of such treatments include reducing agents, protease enzymetreatments, carbohydrate modifications, detergents, urea, andcombinations thereof.

The methods of the present invention can be used to process largevolumes of protein mixtures as compared to the capacities of the ionexchange columns being utilized. In one aspect, for example, the ionexchange column has a protein capacity that is at least the same as thetotal protein in an undiluted protein mixture. In another aspect, theion exchange column has a protein capacity that is at least 5 timessmaller than the total protein in an undiluted protein mixture.Additionally, in one aspect the high concentration protein mixture has aprotein concentration that is at least 10% greater than ion exchangecolumn protein capacity. In another aspect, the high concentrationprotein mixture has a protein concentration that is at least 20% greaterthan the ion exchange column protein capacity.

The ion exchange columns according to aspects of the present inventioncan include anion exchange columns or cation exchange columns.Additionally, a variety of fixed pH values can be used when separating ahigh concentration protein mixture into a bound protein fraction and anunbound protein fraction. For example, in one aspect the fixed pH isfrom about 2.0 to about 10.0. In another aspect, the fixed pH is fromabout 8.0 to about 10.0. In yet another aspect, the fixed pH is fromabout 2.0 to about 6.0.

In another aspect of the present invention, a method for separating aprotein mixture into a bound protein fraction and a flow-through proteinfraction is provided. Such a method can include delivering a proteinmixture through an ion exchange column at a fixed pH and a fixed saltconcentration, where the fixed pH and the fixed salt concentration havebeen preselected to cause separation of the protein mixture into a boundprotein fraction that binds to the ion exchange column and aflow-through protein fraction that flows through the ion exchangecolumn, and where greater than or equal to about 5% of protein in theprotein mixture is in the flow through fraction. The method alsoincludes receiving the flow-through protein fraction from the ionexchange column separate from the bound protein fraction, and whereineither the bound protein fraction or the flow-through fraction containsa protein of interest.

In yet another aspect of the present invention, a method for isolating ahigh abundant protein from a high concentration protein mixture isprovided. Such a method includes delivering a high concentration proteinmixture through an ion exchange column at a fixed pH and a fixed saltconcentration, wherein the fixed pH and the fixed salt concentrationhave been preselected to cause separation of the protein mixture into abound protein fraction and a flow-through protein fraction. The boundprotein fraction binds to the ion exchange column and the flow-thoughprotein fraction flows though the ion exchange column, and, in thiscase, a majority of the high abundant protein is in the bound proteinfraction. The method also includes receiving the flow-through proteinfraction from the ion exchange column and subsequently eluting the boundprotein fraction including the majority of the high abundance proteinfrom the ion exchange column.

A variety of high abundant proteins are contemplated, and it should benoted that any protein that is in high abundance in a protein mixtureshould be seen as being within the present scope. In one aspect,however, non-limiting examples can include transferrin, immunoglobulins,albumin, and combinations thereof.

The fixed pH and fixed salt concentration can vary depending on the highabundance proteins being separated and the nature of the proteinmixture. In one aspect, however, the fixed pH is from about 2.0 to about10.0 and the fixed salt concentration is from about 2 mM to about 400mM. In another aspect, the fixed pH is from about 5.2 to about 8.2 andthe fixed salt concentration is from about 20 mM to about 300 mM.

There has thus been outlined, rather broadly, the more importantfeatures of the invention so that the detailed description thereof thatfollows may be better understood, and so that the present contributionto the art may be better appreciated. Other features of the presentinvention will become clearer from the following detailed description ofthe invention, taken with the accompanying drawings and claims, or maybe learned by the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Additional features and advantages of the invention will be apparentfrom the detailed description which follows, taken in conjunction withthe accompanying drawings, which together illustrate, by way of example,features of the invention.

FIG. 1 is an image showing protein separation after dialysis by1-dimensional SDS gel electrophoresis according to one aspect of thepresent invention;

FIG. 2 is an image showing unbound protein separation after dialysis by2-dimensional gel electrophoresis according to another aspect of thepresent invention;

FIG. 3 is an image showing bound protein separation by 2-dimensional gelelectrophoresis after elution and dialysis according to yet anotheraspect of the present invention; and

FIG. 4 is a graphical representation of bound vs. unbound protein atdifferent pH and salt conditions according to a further aspect of thepresent invention.

DETAILED DESCRIPTION

Before the present invention is disclosed and described, it is to beunderstood that this invention is not limited to the particularstructures, process steps, or materials disclosed herein, but isextended to equivalents thereof as would be recognized by thoseordinarily skilled in the relevant arts. It should also be understoodthat terminology employed herein is used for the purpose of describingparticular embodiments only and is not intended to be limiting.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a protein” includes one or more of such proteins, andreference to “the column” includes reference to one or more of suchcolumns.

DEFINITIONS

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set forthbelow.

As used herein, the term “high abundance protein” refers to proteinsthat make up greater than about 5 wt % of a protein mixture.

As used herein, the term “high concentration protein mixture” refers toa protein mixture that has a protein concentration that is equal to orhigher than the protein capacity of an ion-exchange column through whichsuch a protein mixture is passed.

As used herein, the term “affinity binding” refers to binding between aprotein and a substrate that is not a result of an ion-exchangeinteraction.

As used herein, the term “protein capacity,” when referring to anion-exchange column, refers to the amount of protein that is capable ofbinding to the column.

As used herein, the term “substantially” refers to the complete ornearly complete extent or degree of an action, characteristic, property,state, structure, item, or result. For example, an object that is“substantially” enclosed would mean that the object is either completelyenclosed or nearly completely enclosed. The exact allowable degree ofdeviation from absolute completeness may in some cases depend on thespecific context. However, generally speaking the nearness of completionwill be so as to have the same overall result as if absolute and totalcompletion were obtained. The use of “substantially” is equallyapplicable when used in a negative connotation to refer to the completeor near complete lack of an action, characteristic, property, state,structure, item, or result. For example, a composition that is“substantially free of” particles would either completely lackparticles, or so nearly completely lack particles that the effect wouldbe the same as if it completely lacked particles. In other words, acomposition that is “substantially free of” an ingredient or element maystill actually contain such item as long as there is no measurableeffect thereof.

As used herein, the term “about” is used to provide flexibility to anumerical range endpoint by providing that a given value may be “alittle above” or “a little below” the endpoint.

As used herein, a plurality of items, structural elements, compositionalelements, and/or materials may be presented in a common list forconvenience. However, these lists should be construed as though eachmember of the list is individually identified as a separate and uniquemember. Thus, no individual member of such list should be construed as ade facto equivalent of any other member of the same list solely based ontheir presentation in a common group without indications to thecontrary.

Concentrations, amounts, and other numerical data may be expressed orpresented herein in a range format. It is to be understood that such arange format is used merely for convenience and brevity and thus shouldbe interpreted flexibly to include not only the numerical valuesexplicitly recited as the limits of the range, but also to include allthe individual numerical values or sub-ranges encompassed within thatrange as if each numerical value and sub-range is explicitly recited. Asan illustration, a numerical range of “about 1 to about 5” should beinterpreted to include not only the explicitly recited values of about 1to about 5, but also include individual values and sub-ranges within theindicated range. Thus, included in this numerical range are individualvalues such as 2, 3, and 4 and sub-ranges such as from 1-3, from 2-4,and from 3-5, etc., as well as 1, 2, 3, 4, and 5, individually. Thissame principle applies to ranges reciting only one numerical value as aminimum or a maximum. Furthermore, such an interpretation should applyregardless of the breadth of the range or the characteristics beingdescribed.

Invention

In the practice of protein ion-exchange chromatography, proteins can beeluted from an ion-exchange column by an increase in salt concentration(e.g. NaCl) or a change in pH. In a typical procedure, a protein mixtureis run through a column such that essentially all of the protein in themixture is bound to the column. In such cases, only small amounts of aprotein mixture can be processed to allow all of the protein in thesample to bind to the column. Binding is followed by an increase in saltconcentration or a decrease in pH to elute the proteins from the column.The increase in salt or decrease in pH is usually performed as agradient change to allow sufficient protein separation to occur. Thisprotein separation scheme can be hampered, however, by the presence ofhigh abundance proteins such as albumin, transferrin, IgG, and the like.These high abundance proteins can impede the separation and/or theidentification of low abundance proteins from the protein mixture.

The inventor has discovered that proteins from a protein mixture can beseparated without having total binding of all of the proteins in aprotein mixture to the column. In this case, proteins are separated intoa bound fraction that binds to the column and a flow-through fractionthat flows though the column prior to elution. As such, this processproduces a meaningful separation of proteins, namely, the proteins areseparated into bound and flow-through fractions as compared totraditional separation processes whereby essentially all of the proteinin a protein sample is bound to the column and salt and/or pH gradientscause protein separation as proteins are eluted off the column. As such,one significant benefit of the methods according to aspects of thepresent invention pertains to the amount of protein that can beseparated. In this case, high abundance proteins can be separated fromlower abundance proteins in much higher concentrations of proteinmixtures than is possible with traditional ion-exchange methods.

Accordingly, in one aspect, a method for separating a high concentrationprotein mixture into a bound protein fraction and a flow-through proteinfraction is provided. Such a method includes delivering a proteinmixture through an ion exchange column at a fixed pH and a fixed saltconcentration, wherein the fixed pH and the fixed salt concentrationhave been preselected to cause separation of the protein mixture into abound protein fraction and a flow-through protein fraction. Thus thebound protein fraction binds to the ion exchange column and theflow-though protein fraction flows though the ion exchange column suchthat the flow-through protein fraction is received and collected as itflows through the ion exchange column while the bound protein fractionremains bound to the column. The bound protein fraction is then elutedfrom the column and is received and collected separately from theflow-through protein fraction. Additionally, either the bound proteinfraction or the flow-through fraction can contain a protein of interest.It should be noted that, in one aspect, the present techniques encompassion-exchange binding of protein to adsorbent, and that such binding isnot a result of affinity binding.

In another aspect of the present invention, a method for separating aprotein mixture into a bound protein fraction and a flow-through proteinfraction is provided. The protein mixture can contain protein of anyconcentration, including those protein mixtures defined as highconcentration protein mixtures. The method includes delivering a proteinmixture through an ion exchange column at a fixed pH and a fixed saltconcentration, where the fixed pH and the fixed salt concentration havebeen preselected to cause separation of the protein mixture into a boundprotein fraction that binds to the ion exchange column and aflow-through protein fraction that flows through the ion exchangecolumn, and where greater than or equal to about 5% of protein in theprotein mixture is in the flow through fraction. The method alsoincludes receiving the flow-through protein fraction from the ionexchange column and subsequently eluting and receiving the bound proteinfraction separately from the flow-through fraction. A protein ofinterest can be contained either the bound protein fraction or theflow-through fraction. In another aspect, greater than or equal to about25% of protein in the protein mixture is in the flow through fraction.In yet another aspect, greater than or equal to about 50% of protein inthe protein mixture is in the flow through fraction. In a furtheraspect, greater than or equal to about 75% of protein in the proteinmixture is in the flow through fraction.

Many of the high abundance proteins in a typical serum/plasma mixturehave high isoelectric points (pI) and/or low affinities toanion-exchange absorbents. Standard chromatography protocols dictatethat conditions should be set for all proteins to bind to the column,thus necessitating higher pH conditions (e.g. >7.0). As an example,immunoglobulin proteins generally have isoelectric points above pH 6.5.As such, a majority of immunoglobulins can be made to flow through thecolumn by lowering the buffering conditions on the column and of theprotein mixture to below pH 6.5. In such a situation, the majority ofimmunoglobulins will not bind to the column and will thus be present inthe flow-through fraction. Processing such a protein mixture at evenlower pH conditions will lower the affinity of immunoglobulins to thecolumn even lower, thus increasing the amount of immunoglobulin thatpasses through the column in the flow-through fraction.

As another example, albumin is a high abundance protein that can passthrough an ion-exchange column under the proper conditions. Althoughalbumin has a pI of about pH 4.5, this protein does not bind well toanion-exchange columns, possibly due to structural constrains of suchproteins. As one non-limiting example, lowering the bufferingconcentration conditions of the column and of the protein mixture toabout pH 6.2 in about 20-150 mM NaCl will allow a majority of albuminproteins to flow through the column. It should be noted that albumin,transferrin, and immunoglobulin proteins are categorizations that canrepresent different protein species. The pI properties of immunoglobulinproteins made by different B-cells create variability. Albumin bindsmany different small molecules that create pI variability andcolumn-binding characteristics as well. Additionally, transferrin candisplay different column affinity depending on the presence or absenceof iron molecules.

By setting column and protein mixture conditions to a pH that is lowerthan 6.5, and increasing the NaCl concentration, the high abundanceproteins (e.g. immunoglobulin, transferrin, albumin, fibrinogen, and thelike) can be separated from lower abundance proteins on smallion-exchange columns. Surprisingly large volumes of protein can beprocessed using such small ion-exchange columns. For example, ananion-exchange column containing about 100 μl of DEAE-cellulose canallow for the processing of greater than 1 ml of undiluted serum proteinmixture such that the low abundance proteins bind to the column and thehigh abundance proteins flow through the column. If a protein ofinterest is in the bound protein fraction, the column can be eluted bylowering pH further or by increasing the salt concentration.

If, for example, the high abundance proteins made up 75% of the proteinsin the protein mixture, and conditions were set so that the highabundance proteins flow through, then only about 25% of proteins in themixture will bind to the column. Thus if the conditions are set to causethe high abundance proteins to flow through, and the remaining proteinsin the protein mixture are less than the binding capacity of the column,then substantially all of these remaining proteins should bind to thecolumn, provided they are capable of binding given the conditions. Ifthere are more remaining proteins in the protein mixture than thebinding capacity of the column, then a portion of the remaining proteinswill flow through the column as the binding capacity is reached.

In another aspect, a weak or strong cation-exchange absorbant can beused. Non-limiting examples include CM-cellulose, sulfoethyl cellulose,and the like. Additionally, more rigid agarose or polymeric adsorbentscan also be used. The use of cation-exchange adsobants generally isbenefited from the use of lower pH buffering conditions (e.g. from aboutpH 4.5 to about pH 6.0) in low salt conditions (e.g. from about 5 mM toabout 154 mM). At these conditions, proteins with a high pI such asimmunoglobulins, transferrin, fibrinogen, and the like, bind with highaffinity to the negatively charged adsorbent. This thus allows for thebinding of high abundance proteins to the column and the collection ofunbound low abundance proteins from the flow-through fraction. Onebenefit of such a technique may be the further separation of proteinspreviously separated using an anion-exchange process, or if a protein ofinterest has a high pI.

Various protein mixtures are contemplated for separation according tothe aspects of the present invention, and any protein mixture capable ofsuch separation should be considered to be within the present scope. Inone aspect, for example, the protein mixture can be a biological fluid.Non-limiting examples of biological fluids can include blood serum,blood plasma, urine, CNS fluid, saliva, cellular extracts, tissueculture extracts, and the like. In one specific aspect, the biologicalfluid can be serum or plasma.

One benefit of the present techniques for protein separation is theability to process protein mixtures via ion-exchange chromatography thatcontain high concentrations of protein. In many aspects, for example,the protein mixture can be an undiluted biological fluid. In traditionalion-exchange procedures, a protein sample is heavily diluted in order toreduce the protein concentration in the sample to a level that allowsall of the protein to bind to the column. The present methods allow anundiluted protein mixture, such as an undiluted biological fluid, to beprocessed via ion-exchange chromatography in order to ascertain thepresence of a protein of interest, separate high abundance proteins,etc. It should be noted that the addition of column buffer to a proteinmixture should not necessarily be considered dilution. For example, a 5ml whole serum sample processed on a column is not considered “diluted”by the addition of 5 ml of column buffer to facilitate movement throughthe column because 5 ml of serum is still loaded on the columnregardless of the buffer. It should also be noted that in some aspectsthe protein mixture can be diluted to lower the salt concentration tolevels below normal saline (154 mM NaCl).

It can be beneficial, however, to remove from biological fluids tissue,cells, or other large biological matter that can hamper the ion-exchangeprocess. Biological fluids derived from blood, for example, can beallowed to clot to remove blood cells therefrom. It should be noted,however, that the present scope should not be limited to undilutedbiological fluids, and that some level of dilution may be beneficial,depending on the specific ion-exchange conditions and the proteins beingseparated.

In another aspect of the present invention, the protein mixture can be anon-dialyzed biological fluid. Protein mixtures can be processedaccording to the present techniques in a non-dialyzed, a substantiallynon-dialyzed, or a dialyzed state. Traditional approaches toion-exchange chromatography exhaustively dialyze a biological samplesuch as serum prior to processing. The present techniques allow abiological fluid to be processed at physiological salinity levels. Ofcourse, the salinity can be increased or decreased relative tophysiological conditions, depending on the protein of interest and theparticular protocol being performed. It should also be noted that insome aspects the protein mixture can be dialyzed.

Certain procedures are compatible with higher salt concentrations, andin such cases, high salt protein mixtures can be separated andsubsequently processed. Non-limiting examples of such procedures caninclude size exclusion chromatography, immobilized metal affinitychromatography, hydrophobic interaction chromatography, and the like.Certain procedures are facilitated with lower salt concentrations, andas such, flow-through fractions and/or bound fractions can be dialyzedor ultrafiltrated following protein separation. Non-limiting examples ofsuch procedures can include one-dimensional polyacrylamide gelelectrophoresis, other electrophoresis procedures such asiso-electrofocusing, 2-dimensional gel electrophoresis, and the like.

In yet another aspect, the protein mixture can be a non-ultafiltratedbiological fluid. Ultrafiltration is known in the art, and is atechnique that is typically used in traditional ion-exchangechromatography approaches. The present techniques allow the processingof protein mixtures that have not been filtered to such an extent, andthus can allow an increased protein separation performance, as well asdecreased protein separation time. It should be noted, however, that thepresent scope also includes the processing of protein mixtures that havebeen substantially non-ultrafiltrated, as well as those that have beenultrafiltrated. Additionally, as is discussed above, it can bebeneficial to remove cellular material, tissue, and other debris fromthe protein mixture in order to avoid clogging the column. Such aremoval can be accomplished by filtration of the protein mixture, andsuch filtration should not be seen as ultrafiltration.

A protein mixture such as a biological fluid can additionally be treatedprior to or during ion-exchange chromatography in order to modify aprotein or proteins contained therein. Such modifications can be used toenhance the separation procedure, to facilitate future processing stepsto be performed on proteins of interest, to facilitate proteinidentification, and the like. Non-limiting examples of such treatmentscan include reducing agents, protease enzyme treatments, detergentaddition, urea, carbohydrate modifications, and the like.

As has been described, the pH of the column during separation caninfluence the collection of proteins in the flow-through fraction vs.the proteins in the bound fraction. Thus the fixed pH can be within anumber of pH ranges, depending on the desired protein separation, theprotein of interest, and whether or not it is intended for the proteinof interest to be in the bound fraction or the flow-through fraction. Assuch, any fixed pH can be within the present scope, depending on thesefactors. In one aspect, for example, the fixed pH can be from about 2.0to about 10.0. In another aspect, the fixed pH can be from about 8.0 toabout 10.0. In yet another aspect, the fixed pH can be from about 2.0 toabout 6.0.

One useful benefit of the present techniques is the ability to processquantities of protein that are much higher than the traditional loadingcapacities of ion-exchange columns. Thus, because all protein in asample is not required to bind to the column, very high concentrationsof protein in a protein mixture can quickly be separated using thesetechniques. For example, in one aspect, the ion exchange column has aprotein capacity that is at least the same as the total protein in anundiluted protein mixture. As such, a protein mixture having totalprotein content that is at least the same as the protein capacity of agiven ion-exchange column can be processed using the present techniques.In another aspect, the ion exchange column has a protein capacity thatis at least 5 times smaller than the total protein in an undilutedprotein mixture. Similarly, a protein mixture having a total proteincontent that is at least 5 times the protein capacity of a givenion-exchange column can be processed using the present techniques. Inyet another aspect, a high concentration protein mixture has a proteinconcentration that is at least 10% greater than the ion exchange columnprotein capacity. In another aspect, a high concentration proteinmixture has a protein concentration that is at least 20% greater thanthe ion exchange column protein capacity.

As has been described, the present techniques can additionally be usefulfor the removal of high abundance proteins from a protein mixture. Inone aspect, for example, a method for isolating a high abundant proteinfrom a high concentration protein mixture is provided. Such a method caninclude delivering a high concentration protein mixture through an ionexchange column at a fixed pH and a fixed salt concentration, where thefixed pH and the fixed salt concentration have been preselected to causeseparation of the protein mixture into a bound protein fraction and aflow-through protein fraction. In this case, the bound protein fractionbinds to the ion exchange column and the flow-though protein fractionflows though the ion exchange column. Thus, the fixed pH and the fixedsalt concentration are set to allow a majority of the high abundantprotein to be bound to the column. The flow-through fraction can bereceived from the ion exchange column separate from the bound proteinfraction, and the bound protein fraction, including the majority of thehigh abundance protein, can be eluted from the ion exchange column.

The ranges within which the fixed pH and fixed salt concentrations arelocated can vary, depending on the conditions of the separation protocoland the particular high abundance proteins being bound to the column. Inone aspect, for example, the fixed pH is from about 2.0 to about 10.0and the fixed salt concentration is from about 2 mM to about 400 mM. Inanother aspect, the fixed pH is from about 5.2 to about 8.2 and thefixed salt concentration is from about 20 mM to about 200 mM. It shouldbe noted that, in one aspect, the ion-exchange column is a cationexchange column. Similarly, in some aspects the ion-exchange column canbe an anion exchange column. It should also be noted that the boundprotein fraction is bound to the column by ionic charges as opposed toaffinity binding.

EXAMPLES Example 1

Human off clot serum is filtered through a 0.45 micron filter to removeparticulate matter capable of plugging chromatographic columns. ABIO-RAD MACROPREP HIGH Q® column (1 ml) is equilibrated in about 15-20column volumes of 20 mM Na MES, pH 6.2 with 0.154M NaCl (normal saline).8 ml of whole serum is added to 8 ml of 20 mM Na MES, pH6.2 with 0.154MNaCl. The sample mixture is adjusted to pH 6.2 with NaOH. The samplesolution is pumped onto the column at a rate of 0.5 ml/min. The columneffluent is collected as the flow-through unbound fraction. Afterapplication of the sample solution, the column is washed with 5 ml ofcolumn buffer (20 mM Na MES, pH 6.2 with 0.154M NaCl). The boundproteins are then eluted with 5 ml of 1M NaCl and are maintainedseparately from the flow-through fraction. FIG. 1 shows the flow-throughor unbound fraction (left), molecular weight markers (middle), and thebound fraction (right). This figure demonstrates the separation that isachieved using the present techniques, and particularly the absence ofhigh abundance proteins such as IgG, transferrin, and albumin from thebound fraction. FIG. 2 shows 100 ug of total protein from the unboundfraction following processing on a 2D 10% SDS gel.

Example 2

The protocol of Example 1 is repeated to examine the proportion ofprotein bound to a column for a series of fixed pH and fixed NaClconcentrations. Buffers used to equilibrate the column at a fixed pH areshown in Table 1. A series of protein separations are run for each pH atvarying NaCl concentrations, namely, 0.15M NaCl (normal saline), 0.077NNaCl (½ normal saline), 0.035M NaCl (¼ normal saline) and 0.019M (⅛normal saline). The results for this series of experiments are shown inFIG. 4.

TABLE 1 Fixed pH Buffer 3.2 Sodium Formate 4.2 Sodium Citrate 5.2 SodiumAcetate 6.2 Sodium MES 7.2 Sodium Phosphate 8.2 Tris-HCl 9.2 SodiumBorate 10.2 Sodium Beta-alanine

Example 3

The following protocol is used for the removal of high abundanceproteins from a protein mixture using a cation-exchange column.

Human off clot serum is filtered through a 0.45 micron filter to removeparticulate matter capable of plugging chromatographic columns. ABIO-RAD MACROPREP HIGH S® column (1 ml) is equilibrated in about 15-20column volumes of 10 mM sodium citrate buffer (pH 4.2) with 0.019 M NaCl(1/8 normal saline). 0.25 ml of filtered (0.45 micron) serum is mixedwith 0.75 ml of 10 mM sodium citrate buffer, and to the resultingmixture is added 2.0 ml of 10 mM sodium citrate buffer with 0.0195 MNaCL (1/8 normal saline) to form 3.0 ml of column sample. The sample isapplied to the column that has been equilibrated in 10 mM sodium citratebuffer with 1/8 normal saline. The column effluent is collected as theflow-through unbound fraction. The column is washed with 5 ml of 10 mMsodium citrate buffer with 1/8 normal saline, and the bound proteins arethen eluted with 5 ml of 1M NaCl and are maintained separately from theflow-through fraction. The preceding example thus provides effectiveremoval of high abundance proteins from a protein mixture.

Example 4

The following protocol is used for the enrichment of urine proteins byanion-exchange chromatography. Large volumes of urine can be processedbecause protein content is low compared to serum. 50 ml of human urineis added to 5 ml of 20 mM sodium acetate (pH 5.2 adjusted with NaOH) toproduce a 55 ml column sample. A BIO-RAD MACROPREP HIGH Q® column (1 ml)is equilibrated in about 15-20 column volumes of 20 mM sodium acetate(pH 5.2) with 0.077M NaCl (1/2 normal saline). The sample is applied tothe column at a rate of 0.5 ml/min, and the column effluent is collectedas the flow-through fraction. The column is washed with 5 ml of 20 mMsodium acetate (pH 5.2 with 0.077 M NaCl (1/2 normal saline), and thebound proteins are then eluted with 5 ml of 1M NaCl and are maintainedseparately from the flow-through fraction. The preceding example thusprovides effective enrichment of urine proteins.

Of course, it is to be understood that the above-described arrangementsare only illustrative of the application of the principles of thepresent invention. Numerous modifications and alternative arrangementsmay be devised by those skilled in the art without departing from thespirit and scope of the present invention and the appended claims areintended to cover such modifications and arrangements. Thus, while thepresent invention has been described above with particularity and detailin connection with what is presently deemed to be the most practical andpreferred embodiments of the invention, it will be apparent to those ofordinary skill in the art that numerous modifications, including, butnot limited to, variations in size, materials, shape, form, function andmanner of operation, assembly and use may be made without departing fromthe principles and concepts set forth herein.

What is claimed is:
 1. A method for isolating high abundant proteinsfrom a high concentration protein mixture, comprising: delivering a highconcentration protein mixture through an anion exchange column at afixed pH and a fixed salt concentration, wherein the fixed pH and thefixed salt concentration have been preselected to cause separation ofthe protein mixture into a bound protein fraction and a flow-throughprotein fraction, wherein the bound protein fraction binds to the anionexchange column and the flow-through protein fraction flows though theanion exchange column without binding to the column, and wherein amajority of the high abundant proteins are in the flow-through proteinfraction; and receiving the flow-through protein fraction containing themajority of the high abundant proteins from the anion exchange columnseparate from the bound protein fraction.
 2. The method of claim 1,wherein the high abundant proteins include a plurality of proteins fromthe group consisting of transferrin, immunoglobulin, albumin, andfibrinogen.
 3. The method of claim 1, wherein the fixed pH is from about2.0 to about 10.0 and the fixed salt concentration is from about 2 mM toabout 400 mM.
 4. The method of claim 1, wherein the fixed pH is fromabout 5.2 to about 8.2 and the fixed salt concentration is from about 20mM to about 300 mM.
 5. The method of claim 1, wherein the bound proteinfraction does not affinity bind to the anion exchange column.
 6. Themethod of claim 1, wherein the fixed pH is less than or equal to about7.0.
 7. The method of claim 1, wherein the fixed pH is less than orequal to about 6.5.
 8. The method of claim 1, wherein the fixed pH isfrom about 2.0 to about 6.0.
 9. The method of claim 1, furthercomprising eluting the bound protein fraction from the anion exchangecolumn.
 10. The method of claim 1, wherein the high concentrationprotein mixture is a biological fluid.
 11. The method of claim 10,wherein the biological fluid is selected from the group consisting ofblood serum, blood plasma, urine, CNS fluid, saliva, cellular extracts,tissue culture extracts, and mixtures thereof.
 12. The method of claim10, wherein the high concentration protein mixture is an undilutedbiological fluid.
 13. The method of claim 10, wherein the highconcentration protein mixture is a non-dialyzed biological fluid. 14.The method of claim 10, wherein the high concentration protein mixtureis a non-ultafiltrated biological fluid.
 15. The method of claim 1,wherein the anion exchange column has a protein capacity that is atleast the same as the total protein in an undiluted protein mixture. 16.The method of claim 1, wherein the anion exchange column has a proteincapacity that is at least 5 times smaller than the total protein in anundiluted protein mixture.
 17. The method of claim 1, wherein the highconcentration protein mixture has a protein concentration that is atleast 10% greater than the anion exchange column protein capacity. 18.The method of claim 1, wherein the high concentration protein mixturehas a protein concentration that is at least 20% greater than the anionexchange column protein capacity.
 19. The method of claim 1, whereingreater than 50% of protein in the protein mixture is in the flowthrough fraction.
 20. The method of claim 1, wherein greater than orequal to about 75% of protein in the protein mixture is in the flowthrough fraction.